Synergistic Study of Silver Nanoparticles and Antibiotics
Info: 5657 words (23 pages) Dissertation
Published: 24th Feb 2022
Tagged: Biomedical Science
In the post antibiotic era where increasing emergence of multidrug and antibiotic resistant strains of bacteria becomes a global threat, urges the need to discover novel therapeutic methods to enhance drug efficacy and delivery methods. Silver nanoparticle has been explored as one the potential candidate to overcome the antibiotic crisis where functionalized and conjugated silver nanoparticles becomes the focused-on research. One aspect of these research is to study the synergistic effect of silver nanoparticle and antibiotic towards microorganism particularly standard type bacteria and clinically isolated pathogen. Thus this review aims provides insights on the researches done on synergistic study of silver nanoparticle and antibiotic along with the central idea and issues that the researchers would wanted to address.
Mercury and silver compounds are well known for their antimicrobial properties and have been used as germicide in the past. The toxic effects of metal nanoparticles towards eukaryotic and prokaryotic microorganisms are known as the oligodynamic effect (Talaro & Chess, 2018). Usually they are prepared as inorganic or inorganic metallic salt (Talaro & Chess, 2018).
Compared to macro-scaled materials, nano-materials possesses unique physical, chemical, and biological properties where it seems to provide potential solution to challenges faced in the areas of catalysis and advance medicine (Yun & Lee, 2017). Silver nanoparticles in particular have attracted attention from researchers because of their large number of applications which includes drug delivery potentials, nanocomposites, antimicrobial products and filters (Vlăsceanu et al., 2016). Numerous research revealed the advantageous size- and shape-depending properties of AgNPs can be used for such purposes (Vlăsceanu et al., 2016). Due to their antimicrobial activities, silver nanoparticles (AgNP) are often highlighted in modern medical microbiology (Yun & Lee, 2017).
The variety antimicrobial mechanisms that AgNP act upon prokaryotic microorganisms includes membrane disruption, apoptosis and synergy (Yun & Lee, 2017). In eukaryotic fungal cells, the interaction of AgNPs with fungal membrane causes the disruption of its membrane integrity (Yun & Lee, 2017). The cystosol increment of hydroxyl radicals and mitochondrial dysfunction are the crucial antimicrobial properties in silver nanoparticles–induced apoptosis of microorganisms (Yun & Lee, 2017). Moreover, silver nanoparticles are also known to trigger apoptotic cell death in bacteria by DNA fragmentation and phosphatidylserine exposure (Yun & Lee, 2017). Due to their novel properties, silver nanoparticles are also considered an ideal potential for anti-cancer therapy (Vlăsceanu et al., 2016). Numerous recent research have profess that silver nanoparticles may have antitumoral agent potential implicated by their antiproliferative and apoptosis-inducing capabilities (Vlăsceanu et al., 2016).
Antibiotics are antimicrobial drugs that are too often exploited to control infectious diseases. Incidence of rapid increase of antibiotic resistance strains emphasizes the need for more effective methods of drug delivery (Das & Patra, 2017). Therefore, to overcome such problems, novel antimicrobial agents are needed to where clinical pathogens cannot easily develop resistance to.
Among all the method proposed, metal nanoparticles conjugated with conventional drugs seems like a potential effective way to overcome microbial infections and resistance (Aftab Ahmad et al., 2016). Moreover, with such synergistic effect, silver nanoparticles may increase the antibacterial capacity of conventional available drugs which then confronts the emergence of resistance strains and biofilm formations (Yun & Lee, 2017). Functionalised nanomaterials have high potential to combat emerging of resistant strains because their antimicrobial effects are dependent on their abilities to affect multiple biological pathways of pathogens (Das & Patra, 2017). Moreover, to further develop microbial resistance towards functionalised antimicrobial nanoparticles (NPs), concurrent mutations have to develop resistance to nanoparticles which makes an double resistance event unlikely (Das & Patra, 2017).
Silver Nanoparticles (AgNPs)
Silver metal’s antimicrobial properties has been widely capitalised based on its broad spectrum inhibition, oligodynamic action, low possibilities for microbial resistance development (Agnihotri, Mukherji, & Mukherji, 2014). However, it should be known that silver nanoparticles exhibit physical and chemical properties that are different from that of bulk silver and nanoparticles exhibit advanced catalytic and antimicrobial activity (Khatoon, Nageswara Rao, Mohan, Ramanaviciene, & Ramanavicius, 2017). Silver nanoparticles antimicrobial activities lies in their capabilities to impose complex mechanism of action by interfering respiratory chain mechanism, DNA replication and inhibition of cellular protein synthesis (Aftab Ahmad et al., 2016). As a result, nano size range silver particles have been most exploited to be used as nano-antimicrobial additive in consumer’s cosmetic and medical products (Agnihotri et al., 2014).
Studies have demonstrated AgNPs antimicrobial nature to be size and shape dependent, where smaller nanoparticles exhibit better antimicrobial activity (Agnihotri et al., 2014; Morones et al., 2005; Pal, Tak, & Song, 2007). Among all shapes of AgNP, truncated-triangular shaped silver nanoparticles appeared to exhibit more effective antimicrobial killing actions (Agnihotri et al., 2014). However, for practical applications in either colloidal form or immobilized state, spherical nanoparticles are still considered to be the best-suited (Agnihotri, Mukherji, & Mukherji, 2013; Agnihotri et al., 2014).
Although there are significant benefits and potential of using silver nanoparticles as antimicrobial agents, however one should acknowledge the fact that AgNP may produce adverse effects on cells such with the production of reactive oxygen species that are highly toxic to both bacteria and eukaryotic cells (Brown et al., 2012).
Synthesis of Silver Nanoparticles
Although there have been success in synthesizing AgNPs of various dimensions and size ranges, however many reported methods contains certain limitations in terms of shape, size and stability controls in the dispersion system (Agnihotri et al., 2014).
Despite that, borohydride-mediated reduction are the exceptions where it is utilise for the chemical reduction synthesis of dispersible silver nanoparticles (Agnihotri et al., 2014; Steinigeweg & Schlücker, 2012). Indeed there are much difficulty faced by early researchers to synthesise silver nanoparticles below 10 nm in size of high monodispersity and stability (Agnihotri et al., 2014). Therefore, a strong reducing agent such as sodium borohydride (NaBH4) is needed to generate instant nuclei, resulting in the formation of uniformly small sized monodispersed silver nanoparticle (Agnihotri et al., 2014). However, by using sodium borohydride reduction, it is not easy to synthesise large size silver nanoparticles (Agnihotri et al., 2014; Pyatenko, Yamaguchi, & Suzuki, 2007). In contrast, the weaker reducing agent trisodium citrate (TSC) is able to chemically generate fairly large silver nanoparticles with a larger size range (Agnihotri et al., 2014)). However, TSC reduction method will also produce silver nanoparticles with a variety of shape such as the highly desired spherical nanoparticles along with undesired mixture of rods, cubes, and triangles (Agnihotri et al., 2014; Dong et al., 2009; Sharma, Yngard, & Lin, 2009).
Apart from chemically synthesising silver nanoparticles with chemical reduction, there are also eco-friendly ways developed for synthesis of silver nanoparticles which is through natural biological system (Bhainsa & D’souza, 2006). Prime examples of organisms biosynthesizing inorganic materials includes magnetotactic bacteria (known to synthesize magnetite nanoparticles), diatoms (known to synthesize siliceous materials) and S-layer bacteria (known to synthesise calcareous layers and gypsum) (Absar Ahmad et al., 2003; Dickson, 1999; Kröger, Deutzmann, & Sumper, 1999).
Bhainsa & D’souza have investigated the capabilities of eukaryotic fungus Aspergillus fumigatus to extracellular biosynthesise silver nanoparticles (Bhainsa & D’souza, 2006). The Aspergillus fumigatus cell filtrate was used to synthesise silver nanoparticles and it was reported as a fast process were silver ion are produced in minutes (Bhainsa & D’souza, 2006). Apart from Aspergillus fumigatus, the fungus Fusarium oxysporum can also reduce aqueous silver ions to form stable silver hydrosol (Absar Ahmad et al., 2003).
Another eco-friendly way to biosynthesise silver nanoparticles is by using plant extracts have been made popular due to their abundance, availability and broad spectrum bioactive reducing metabolites (Rasheed, Bilal, Iqbal, & Li, 2017). Tropical fruit mangosteen, Garcinia mangostana leaf extract is one of the plant extract capable to reduce aqueous silver ions into silver nanoparticles (Veerasamy et al., 2011). Another prominent culinary herb called mugwort, Artemisia vulgaris leaves extract (AVLE) was also found to be capable of synthesizing silver nanoparticles without any addition of reducing or capping agent (Rasheed et al., 2017).
Besides eukaryotic plants and fungus, prokaryotic microorganisms are capable in synthesizing silver nanoparticles. For instance, Lactobacillus sp bacteria are found to be capable to synthesizing extracellular silver nanoparticles that have antioxidant properties in vitro and in vivo in rats (Dakhil, 2017). In a separate journal, it mentioned that the metabolically versatile actinobacteria Rhodococcus sp. is found to reduce aqueous silver nitrate too where biosynthesis process is governed by the cell enzyme systems (Otari, Patil, Nadaf, Ghosh, & Pawar, 2012).
However, there are points to be considered when biosynthesizing silver nanoparticles is that prokaryotic bacteria and eukaryotic fungi may also synthesise the nanoparticles intracellularly in contrast to synthesizing it extracellularly. In such context, the extraction and purification of silver nanoparticles becomes a challenge to be address.
Synergistic Functionalisation Of Silver Nanoparticle With Antibiotic
As mentioned earlier, the antimicrobial activity of silver nanoparticle (AgNP) depends significantly on the structural morphology and size which it’s activity can be increased synergistically via coating their surface with suitable chemical or natural agents (Kumar et al., 2017). These agents used to cap, coat and functionalized AgNPs may originate from their reduction agent (chemical synthesis of AgNP), from natural compound (bio synthesis of AgNP) or an antimicrobial compound such as antibiotic.
The distinguish use of silver and other metal nanoparticles along with natural antimicrobial compounds such as certain antibiotics and chitosan was concluded widely that different combinations of these two produces synergistic antibacterial effect (Buszewski, Rafiſska, Pomastowski, Walczak, & Rogowska, 2016; S. Chen et al., 2012; S. Chen et al., 2014; Y. Chen et al., 2016). There are various studies that support and indicate that such strategy will enhances the antibacterial properties of metal nanoparticles (Buszewski et al., 2016; S. Chen et al., 2012; S. Chen et al., 2014; Y. Chen et al., 2016).
In a separate study of combining amoxicillin with silver nanoparticles, it is reported that there is greater antibacterial effect on Escherichia coli cells than they were applied separately (Li, Li, Wu, Wu, & Li, 2005). When dynamic test is conducted on the E. coli growth indicates that the exponential and stationary phases of E. coli is greatly reduce and delayed with the presence of AgNP and amoxicillin synergist (Li et al., 2005). In addition, the effect of E. coli preincubated with AgNP is shows that the solutions with more AgNP have more antibacterial effect (Li et al., 2005).
In a particular research conduct using commercial antibiotics with AgNP to combat multidrug resistant clinical Acinetobacter baumannii strain, the combination of AgNPs and either polymyxin B (PMB) or rifampicin exhibit strong synergistic antimicrobial effects towards the multidrug resistant clinical Acinetobacter baumannii strain (Wan et al., 2016). Furthermore, the synergist effect of AgNPs and PMB enhances their individual antimicrobial effect against A. baumannii in their in vivo experiment on mouse, further indicates the potential of applying it on clinical basis for this combination therapy (Wan et al., 2016).
Apart from that all, there was also investigation conducted on whether the B-lactam antibiotic ampicillin synergized to nanoparticles could salvage the efficacy its usage against B-lactam resistant bacteria strains (Brown et al., 2012). This research highlights the importance of antibiotic (ampicillin) funcationalised AgNP to act as potential antibacterial agents with synergistic properties that overcome antibiotic resistance mechanisms of multidrug resistant bacteria strains such as Pseudomonas aeruginosa, Enterobacter aerogenes, and a methicillin-resistant isolate of Staphylococcus aureus (Brown et al., 2012). In a separate similar research conducted by Baker et al., shows a high percentage increased in antibacterial activity when AgNP is functionalized with bacitracin when used against bacitracin resistant strains of Bacillus subtilis, Escherichia coli and Klebsiella pneumoniae. Furthermore, Baker et al., reveals that there are a marked increase in antibacterial properties when AgNP is synergized with antibiotics compared to that of the effect of antibiotic alone among those 3 bacteria mentioned (Baker, Pasha, & Satish, 2015).
Biofilm formation is one of the main causes that contribute to the emergence of drug resistant strains of bacteria. Pathogenic bacterial biofilms shows increase in antibacterial resistance due to the complexity of the inherent architecture within the biofilm community (Habash, Park, Vis, Harris, & Khursigara, 2014). Biofilm provides protection towards the bacteria by limiting the dispersion and penetration of antibacterial compound and thus reduce the efficacy of that compound that normally inhibits planktonic bacteria stage (Habash et al., 2014). There was a particular research conducted on inhibition of biofilm formation by Pseudomonas aeruginosa PAO1 biofilms by assessing the efficacy of citrate-capped silver nanoparticles alone and in synergy with aztreonam (monobactam antibiotic) (Habash et al., 2014). The synergistic antibacterial effect of AgNPs and aztreonam may be due to its penetration capabilities of AgNPs into P. aeruginosa biofilm matrix where the aztreonam deleterious effect act against the bacteria cell envelope (Habash et al., 2014). Similarly, the antibiotic kanamycin synergised with AgNPs also produces combined antibacterial effect on planktonic Pseudomonas aeruginosa (Buszewski et al., 2016).
We look into the simplest experiment comparing the antibacterial effect of using silver nanoparticle alone and compared that to that with a functionalised silver nanoparticle. In Buszewski et al., experiment, the silver nanoparticles are synthesized biologically utilizing the bacteria strain Actinomycete CGG 11n and functionalized it with the antibiotic tetracycline. Data of well-diffussion, MIC and MBC assay from Buszewski et al., indicated that P. aeruginosa (ATCC1014), E. coli (ATCC8739), K. pneumoniae (ATCC700603), S. aureus (ATCC6338) and S. epidermidis (Centre for Modern Interdisciplinary Technologies Nicolaus Copernicus University in Torun) bacteria strains are more susceptible towards the microbial action of tetracycline functionalized silver nanoparticle than that of biosynthesized silver nanoparticles alone. The inhibition zone difference is at least 2mm larger with the minimal inhibitory and bactericidal concentration (MIC and MBC) concentration needed for inhibition and bactericidal is at least twice lower for Tetracycline functionalized AgNP compared to that of biosynthesized AgNP alone. Buszewski et al., showed that overall there are significant increases in the zones of inhibition, MIC and MBC among all 4 bacteria strains when tetracycline functionalized AgNP is tested against biosynthesized AgNP alone.
In a separate experiment, Li et al., demonstrates the individual and combine effect of chemically synthesised silver nanoparticles (AgNP) and amoxiciilin towards the dyanamics of E. coli (strain F220) growth in liquid LB medium indicated by measurement of OD 600 nm. The LB medium was first supplemented with 5 × 106 CFU of E. coli bacteria in 0.150 mg ml−1 amoxicillin and 5 μg ml−1 silver nanoparticle, separately or combined. When added separately, 5 μg ml-1 silver nanoparticle has no significant effect on E. coli growth whereas 0.150mg ml−1 antibiotic amoxicillin induced a slight delay of E. coli bacteria’s exponential growth and stationary phase. In comparison when amoxicillin is synergized with AgNP, the synergistic effect did not only delay the exponential phase significantly moreover it decrease the exponential phase growth duration of E. coli. Li et al., has demonstrated clearly by combining amoxicillin and AgNP result in a greater antibacterial effect on E. coli than either AgNP or amoxicillin applied alone.
Both Buszewski et al., and Li et al., have demonstrated the antibacterial effect of silver nanoparticles and antibiotic alone and compared to that of an antibiotic functionalized silver nanoparticle which provide solid evidence on the synergistic effect of functionalizing silver nanoparticles with antibiotic. Both group of researchers shows evidently that regardless it is chemically or biologically synthesized AgNP, both have antibacterial effect and can be enhance with the presence of a conjugated antibiotic reagent. However, Buszewski et al., and Li et al., did not consider the part of experimenting with antibiotic functionalized AgNP with bacteria strains that are resistance towards the antibiotic functionalized.
To address the issue of synergistic effect of silver nanoparticles (AgNP) on multidrug resistant bacteria strains, Wan et al., combined AgNP with commercial antibiotics to treat carbapenem-resistant Acinetobacter baumannii (aba1604) which aims to determine the synergistic or addictive effect of functionalized AgNP in vitro. Polimyxin B (PMB), rifampicin, and tigecycline are selected as the experimental candidate as these 3 antibiotics are commonly used to treat multi drug resistant A. baumannii along with other antibiotics. Wan et al., uses fractional inhibitory concentration (FIC) method to determine whether the AgNPs and the 3 antibiotic combinations have either antagonistic, additive or synergistic effect based on FIC index (P) values.
(Wan et al., 2016)
FIC index values larger than 4.0 indicate antagonistic effects. FIC index values between 0.5 and 4.0 indicate additive effects. FIC index values lower than 0.5 indicate synergistic effects. Wan et al., experiment results showed that PMB and rifampicin exhibit synergistic effect (P < 0.5) with AgNPs, while tigecycline did not exhibit synergistic effect (P >0.5) but additive effect (0.5 < P < 4.0) with AgNPs. Wan et al., experimental methods and results provide insights to us that although silver nanoparticles and antibiotic combination has overall greater antibacterial effect than either reagent alone, however not all antibiotic combination with AgNP are synergistic, some may be additive and even antagonistic. The FIC index mentioned in Wan et al., journal pointed out a good reference point to investigate the synergistic effect of AgNP with antibiotic.
Apart from that, there are researchers conducting experiment to determine whether silver nanoparticle (AgNP) can be used to salvage the efficacy of an antibiotic when a particular bacterium develops resistance against it. Brown et al., conducted an experiment to determine whether the B-lactam antibiotic ampicillin synergized to nanoparticles could salvage the efficacy its usage against B-lactam resistant bacteria strains. The bacteria used in Brown et al., experiment was the differentiated into ampicillin sensitive strains and ampicillin resistant strains. The ampicillin sensitive strains were Shiga toxin-producing pathogenic E. coli (STEC) serotypes O91:H21 and O157:H7, E. coli K-12 sub-strain DH5α and Vibrio cholerae (TRH7000). The ampicillin drug resistant strains used are Methicillin Resistant Staphylococcus aureus (MRSA), E. aerogenes, P. aeruginosa, and E. coli K-12 substrain DH5α (pPCR-Script Amp SK+) where all of them produces the B-lactamase enzymes. Brown et al. demonstrated that the silver nanoparticles (AgNP) even without the presence of ampicillin is able manage to mount antimicrobial activity towards both Gram-positive and Gram-negative bacteria. It is found that the MBC of AgNP was approximately 4µg/mL where most bacteria strains including multiple-drug-resistant strains are fully susceptible at this concentration. However, Brown et al., stated that V. cholerae able to persist and grow under the concentration of 4µg/mL of AgNP and only susceptible at the concentration of 20µg/mL which is 5 folds higher. As for the antibacterial assay with AgNP functionalized with ampicillin, MBC for most ampicillin resistant and non-resistant bacteria was at 1µg/ml with exception ampicillin resistant V. cholerae where it was at 2µg/ml. However, this amount was 10 times less than the amount of AgNP alone needed for the effective killing of Vibrio cholerae. From this experiment we can conclude that synergistic effect of AgNP with ampicillin (AMP) had increased bactericidal activity of AgNP alone. Brown et al., data suggested that the antibacterial activity of functionalized AgNP-AMP is due to the AMP mounted on the surface of the silver nanoparticle. However, Brown et al., made no attempt to bind ampicillin to AgNP, where they suggested that the binding of amoxicillin to the silver nanoparticle occurred and was responsible for the observe synergistic effect. Brown et al., experiment indicated that silver nanoparticle (AgNP) can be used to salvage the efficacy of an antibiotic when a particular bacterium develops resistance against it and the susceptibility of bacteria towards AgNP and functionalized AgNP is species and strain specific.
Bacteria biofilm have provided a safe haven that shielded bacteria from the antibacterial effect of antimicrobial compound. Habash et al., conducted experiment to determine whether can silver nanoparticles synergized with aztreonam against Pseudomonas aeruginosa PAO1 biofilms. According to Habash et al., P. aeruginosa PAO1 biofilms was challenged with the antimicrobial properties AgNPs alone and in combination with aztreonam. Minimum biofilm eradication concentration (MBEC) high-throughput assay was conducted to determine the antimicrobial efficacy of the mentioned reagent against the biofilms. It was found that the silver nanoparticle and aztreonam alone manage to inhibit planktonic cells growth. Silver nanoparticle alone can reduce biofilm biomass and viability; however, it is not true for aztreonam alone as it not reduces the biofilm biomass accumulation but enhances it’s accumulation. Only when the combinations of silver nanoparticles with aztreonam will then biofilm biomass are inhibited, and viability reduced. FIC Index was used in Habash et al., experiment to determine the specific antibacterial interaction of the AgNP aztreonam combination and found the composition to have synergistic antibacterial effect towards Pseudomonas aeruginosa PAO1 biofilms. Negatively stained transmission electron conducted on P. aeruginosa PAO1 biofilms confirms that the AgNP has penetrated the biofilm has possibly deliver along the antibiotic aztreonam which in combination causes bactericidal action. Through Habash et al., experiment we could see that indeed functionalized AgNP can be potentially used as a drug delivery method to deliver antibiotic payload in to biofilms.
Based on the discussion above, synergized silver nanoparticle with antibiotic could be a potential method to combat the emerging rise of multidrug and antibiotic resistant bacteria strain. It has shown that antibiotic functionalized silver nanoparticle can have synergistic antibacterial effect on not only planktonic resistant and non-resistant bacteria strain but also on biofilm bacteria community. In general, all the mentioned conjugated and functionalized silver nanoparticles properties encouraged and attract more research being conducted for it to be used a potential drug specific targeting and delivery system along with enhancement of drug antibacterial properties. Further studies should be conducted to ensure the in vitro antibacterial properties of functionalized silver nanoparticle could be translated into in vivo applications. Thus, for now functionalized silver nanoparticles with respect to synergistic effect of silver nanoparticle and antibiotic remains as one of the novel crucial research conducted in the post-antibiotic era.
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